The invention addresses a method of ultrasonic detection and localization of contrast agent microbubbles, which method comprises the steps of:
scanning an anatomic region in which the presence of contrast agent microbubbles is or may be foreseen, by transmitting one or more ultrasonic pulses at a first predetermined frequency in said anatomic region;
receiving the reflected ultrasonic signals resulting from the transmit pulses;
identifying the presence of reflected ultrasonic signals having at least one second frequency corresponding to at least the second harmonic of the first fundamental frequency of the ultrasonic transmit signals;
associating said reflected ultrasonic signals having at least one second frequency corresponding to at least the second harmonic of the first fundamental frequency of the ultrasonic transmit signals, to contrast agent microbubbles, acting as reflectors;
determining the position of said microbubbles in the anatomic region according to the time localization of the reflected ultrasonic signal or parts of such reflected ultrasonic signal at said at least one second frequency corresponding to the second harmonic of the first fundamental frequency of the ultrasonic transmit pulses within the duration of the whole reflected ultrasonic signal.
The detection of reflected ultrasonic signals in the frequency range corresponding to the second harmonic of the fundamental frequency of one or more ultrasonic transmit pulses is known in the field of ultrasonic imaging as Harmonic Imaging. The use of contrast agents composed of microbubbles, having the function of ultrasonic pulse reflectors is also known. Microbubbles act as non linear reflectors, whereby the reflected ultrasonic wave has a frequency in the frequency range of the second harmonic of the fundamental frequency of the incident ultrasonic wave or pulse. This allows to recognize the presence of contrast agents in an anatomic region under examination. In fact, stationary tissues have reflectors with a mainly linear behavior, whereby the reflected signals have the highest strength in the frequency range corresponding to the fundamental frequency of the excitation pulse/s. Therefore, the non-linear reflector effect of contrast agent microbubbles is typically used to highlight vascular or lymphatic flows which are not sufficiently echogenic and might not be visible by using conventional imaging, or might be covered with signals reflected from the static tissues of the relevant anatomic region, such as the walls of blood or lymphatic vessels or other tissues.
Harmonic Imaging provides excellent results when contrast agents are present in considerable amounts in tissues and particularly in vascular or lymphatic flows.
However, since the so-called echogenic or static tissues have a non linear behavior besides the linear reflection behavior, the signals reflected from such tissues also have spectral components at the second harmonic of the fundamental frequency of the ultrasonic signal transmitted in the relevant anatomic region. Furthermore, non linear reflection responses of such type may be also generated by micromovements of tissues. The components of reflected signals at frequencies other than the fundamental frequency of the signal transmitted in the anatomic region are typically of lower strength than reflected signals generated by contrast agent microbubbles when said contrast agents are present in considerable amounts. This essentially occurs in large vessels.
In small vessels, such as capillaries or the like, the number of contrast agent microbubbles is very small and may even be as small as one microbubble or a small microbubble population, of the order of one or a few tenths of microbubbles.
When a single contrast agent microbubble or a small contrast agent microbubble population is present, the reflected signal particularly at said second harmonic of the fundamental frequency of the transmit pulse, i.e. the signal transmitted to the body being examined, has a very low strength, which is generally lower than the strength of the contribution to said second harmonic frequency generated by the non linear behavior of the static or echogenic tissue.
Therefore, conventional Harmonic Imaging techniques cannot generally detect the presence of a single microbubble or a small microbubble population in the relevant anatomic region, as conventional Harmonic Imaging does not allow to discriminate between said spectral component of the reflected signal, generated by the non linear behavior of the tissue, and the same spectral component generated by the presence of one microbubble or a small microbubble population.
The detection of single bubbles or small contrast agent microbubble populations is important both for checking tissue vascularization conditions, e.g. for angiographic analyses, and for identifying any microvessel or microcapillary feeding tumor tissues, the latter being characterized by an increased vascularization.
In addition to simple contrast agent microbubble detection, information must be also collected about the localization thereof in the relevant anatomic region.
The invention has the object of providing a method as described hereinbefore, which allows detection and localization of single microbubbles or small microbubble populations, i.e. small numbers of contrast agent microbubbles.
The invention achieves the above purposes by providing a method as described above, which has the following additional steps:
reflected signals are projected in one or more multidimensional spaces, to highlight the evolution of the reflected signal spectrum with time and/or the phase relationships between reflected signal components having different frequencies or frequency ranges, particularly the signal components at the fundamental frequency of the transmit pulse/s and at the second harmonic of the transmit pulse/s;
sample reflected ultrasonic signals are detected, by transmitting ultrasonic pulses to known tissue samples containing no single microbubble or small microbubble population and on known tissue samples containing a single bubble or a small bubble population;
the sample reflected ultrasonic signals are projected in the same multidimensional space to highlight the evolution of the reflected signal spectrum with time and/or the phase relationships between the reflected signal components having different frequencies or frequency ranges;
the projections of the sample reflected ultrasonic pulses for simple tissue and tissue having a single microbubble or a small microbubble population in the multidimensional spaces are compared and unique characteristics are defined for said projections for the simple tissue and the tissue having a single microbubble or a small microbubble population;
the projections of reflected signals in multidimensional spaces are analyzed to identify said diversifying characteristics defined on the basis of the comparison between the projections of sample reflected ultrasonic signals in the multidimensional spaces;
a projection of the reflected signal in said multidimensional space being defined as deriving from a single microbubble or a small microbubble population when it has the characteristics of the projection of the sample reflected ultrasonic signal in said multidimensional space relating to the known sample of tissue having a single microbubble or a small microbubble population.
Regarding the localization within the relevant anatomic region, the above method includes the following additional steps:
scanning the relevant anatomic region by transmitting at least one ultrasonic transmit pulse in said region along a plurality of adjacent scan lines;
receiving the reflected signal along each of said scan lines;
analyzing the reflected signal, with the above described method, along each scan line, to identify one signal component deriving from the presence of one microbubble or a small microbubble population and to identify said component of the reflected signal;
determining the position of the microbubble or the small microbubble population along the corresponding scan line according to the time localization of said component within the duration of the reflected signal;
the position of the microbubble or the small microbubble population in the relevant anatomic region being defined by the position of the scan line and the position of the microbubble or the small microbubble population along the scan line.
According to a first embodiment, a first multidimensional projection is provided by analyzing the reflected signal by means of a Higher Order Spectrum, i.e. HOS (see: Mendel J M. Tutorial on higher-order statistics (spectra) in signal processing and system theory: theoretical results and some applications. Proc.IEEE, 79, 3, 278-305)
Amongst the various HOS techniques or polyspectra, the reflected signal is represented by a so-called bispectrum.
As explained hereafter, the bispectrum is a representation of the reflected signal in a three-dimensional space, which highlights the phase relationships between the spectral components of the reflected signal. A more detailed description of bispectra, as well as a relevant bibliography, will be provided hereafter.
In this case, the reflected signals at the second harmonic of the transmit pulse/s (which is the typical frequency of reflected signals generated by non linear reflectors such as contrast agent microbubbles) have different characteristics depending on whether said reflected signals are generated by the simple tissue of a relevant anatomic district (i.e. having no contrast agent microbubble), or by the tissue of said anatomic region and by one microbubble or a small microbubble population therein.
The representation of the reflected signal -by a bispectrum allows to detect a single microbubble or a small microbubble population by analyzing the characteristics of the bispectrum of said reflected signal.
A preferred method consists in generating the bispectra of reflected signals of ultrasonic pulses transmitted on known tissue samples having no microbubble and on tissue samples having one microbubble or a small microbubble population, thereby obtaining sample signal bispectra which allow to determine whether one microbubble or a small microbubble population is present by simply comparing the sample signal bispectra and the bispectra of the signal reflected from the relevant anatomic region.
The comparison may be performed by analytical mathematical instruments, which extract the typical characteristics of bispectra indicating the presence of one microbubble or a small microbubble population.
Alternatively, by generating a database of sample signal bispectra, containing the characteristics of the bispectrum of a reflected signal indicating the presence or absence of one microbubble or a small microbubble population in the tissue of the relevant anatomic region, an image, e.g. a digital image, of said bispectra may be generated, and the characteristics of said bispectra may be determined by automatic Image Pattern Recognition systems.
Many Image Pattern Recognition systems are known. Amongst these, some use the predictive functions of artificial neural networks or cellular neural networks. Some of the leading edge systems are disclosed in WO2005/020132A1, U.S. Pat. No 5,140,670, EP 1,345,145.
These HOS and particularly the bispectrum are theoretically deemed to be able to highlight the phase relationships between the spectral components of a signal and to better show the nature of said components possibly as regards the source that generated them.
Since the reflected signal changes with time, the use of a bispectrum, which is a static representation thereof at a certain time requires said signal to be made at least quasi static. Furthermore, in the reflected ultrasonic signal, the time localization of signal components with reference to the duration of the signal is particularly relevant for its being related to the reflector position along the signal propagation axis of the axis of view. In ultrasonic imaging, time is known to be equivalent to a measurement of depth or distance from the receiver, therefore time localization of the reflected signal component indicating the presence of one microbubble or a microbubble population is important because such time localization is also a measurement of the reflector position, i.e. of the microbubble or small microbubble population along the axis of view or scan line or along the reflected signal propagation axis.
For the above reasons, according to the invention, when using polyspectra or HOS the reflected signal is divided into a sequence of segments, so-called blocks, each corresponding to a fraction of the overall duration of the reflected signal.
The blocks may be also defined in such a manner that they can overlap at least partially. Thus, a bispectrum is generated for the part of the reflected signal related to each block, and the characteristics thereof are determined as described above to assess whether one microbubble or a small microbubble population is present or absent therein. Each block, as well as its time location with reference to the overall duration of the signal, acts as a position indicator along the axis of view, or scan or propagation of the reflected signal. Therefore, the time length of each block is a measurement of spatial length and may be changed in such a manner as to have a higher or lower time resolution and, as a result, a higher or lower space resolution.
Therefore, as mentioned above, the receive signal is first divided into a succession of segments, or blocks, having a predetermined time length, and predetermined start and end times with reference to the overall duration of the receive signal and then projected on the multidimensional space, a corresponding bispectrum being generated for each signal block. The bispectrum of each signal block is then subjected to steps of extraction of peculiar characteristics corresponding to the absence or presence of one microbubble or a small microbubble population according to the above steps.
In a variant embodiment of invention, methods of time-frequency analysis of the reflected signal are used to determine whether or not a single microbubble or a small microbubble population is present in the tissue under examination.
Various methods exist of time-frequency analysis of a signal (see: Qian, Shie, Introduction to Time-Frequency and Wavelet Transforms, 1st Ed, Prentice Hall PTR, ISBN: 0130303607). Once again the signal is projected in a multidimensional space.
The signal processed by a time-frequency analysis method is then represented in a diagram in which frequency is plotted against time. Once more, receive signals are used which are generated by ultrasonic pulses transmitted on known tissue samples having no microbubbles or just one microbubble or a small microbubble population and the characteristics of the spectrum representation over time obtained with the time-frequency analysis method are identified for signals received from simple tissue and for signals received from tissue having one microbubble or a small microbubble population respectively.
After identification of the characteristics differentiating the time-frequency analysis representations of the receive signal of the two above mentioned types of receive signals, such characteristics may be extracted by comparison or other techniques such as Image Pattern Recognition techniques, from the receive signal of an anatomic region under examination, exactly as it was provided when bispectra were used as a method of multidimensional projection of the receive signal.
In a first embodiment, the so-called Gabor expansion is used (see: Feichtinger & Strohmer (Eds.), GABOR ANALYSIS & ALGORITHMS: Theory & Applications, Birkhauser/SPRINGER-VERLAG, ISBN: 0817639594; Qian, Shie, Introduction to Time-Frequency and Wavelet Transforms, 1st Ed, Prentice Hall PTR, ISBN: 0130303607).
This specific time-frequency analysis of the receive signal allows to recognize and discriminate the presence of one microbubble or a small microbubble population when the spectral component at the second harmonic of the fundamental frequency of the transmit pulse is dominant with respect to the same spectral component of the receive signal generated by a non linear reflection behavior of the tissues in the anatomic region under examination.
If the signal component generated by the presence of a single microbubble or a small microbubble population has a lower or much lower strength than the same spectral component of the receive signal, generated by non linear reflection from tissues, then Gabor expansion does not allow to detect with a relatively high level of accuracy the presence of one microbubble or a small microbubble population.
Another embodiment of time-frequency analysis is the Wigner-Ville distribution in one of its many variants. (see: 0817639594; Qian, Shie, Introduction to Time-Frequency and Wavelet Transforms, 1st Ed, Prentice Hall PTR, ISBN: 0130303607)
Advantages have particularly resulted from using the so-called Pseudo Wigner-Ville Distribution (PWVD).
This type of time-frequency analysis provides acceptable results even when the spectral component of the receive signal resulting from the single microbubble or a small microbubble population has a lower strength than the same spectral component of the signal resulting from non linear reflection from the tissues of the anatomic region under examination.
As explained below in a more detailed discussion of the mathematical formalism of the PWVD, this type of time-frequency analysis seems to not only highlight the evolution of the received signal with time, but also the phase relationships between the spectral components of the received signal.
When compared with the bispectrum, the PWVD provides automatic segmentation of the receive signal into successive time blocks, and also highlights the phase relationships between the spectral components of the receive signal.
According to an improvement, since the spectral component of the receive signal at the frequency corresponding to the fundamental frequency of the transmit pulse has a considerably higher strength than the spectral components of the receive signal at the second harmonic frequency, regardless of whether these result from one or more contrast agent microbubbles, advantages are obtained by providing an additional step before projecting the receive signal in the multidimensional space and/or dividing it into a succession of time blocks. This additional step consists in subjecting the receive signal to filtering or processing to remove the spectral component thereof in the range of the fundamental frequency of the transmit pulse.
The additional filtering step allows to reduce the differences in strength between the remaining signal components at the second or higher harmonic frequency and thereby improves prediction accuracy, i.e. allows to safely determine whether a single microbubble or a small microbubble population is present or not. As stated above, the determination of the presence and position of a single contrast agent microbubble or a small contrast agent microbubble population is considerably important, not only for checking for small vessels but also in combination with new types of contrast agents, in which microbubbles are designed to only bond to target regions or tissue types. In this case, microbubbles have such a chemical and molecular structure as to bond to a desired tissue type.
For instance, in microvessel density measurement, which may be of help in evaluating the presence and the evolution stage of prostate cancer, microbubbles may have bioconjugate ligands which are capable of bonding to the endothelium of the new vessels associated to the vascularization caused by the onset of such tumor. Contrast agent microbubbles having structures with bioconjugate ligands capable of selectively bonding to specific types of derived tissues are known, for instance, from Dayton P., Ferrara K., “Targeted Imaging Using Ultrasound” Journal of Magnetic Resonance Imaging, vol. 16, pp. 362-377, 2002; Hall C. S., Marsh J. N., Scott M. J., Gaffney P. J., et al., “Temperature dependance of ultrasonic enhancement with a site-targeted contrast agent”, Journal of Acoustic Society of America, vol. 110, issue 3, pp. 1677-1684, September 2001; Hughes M. S.,m Lanza G. M., Marsh J. N. Wickline S. A., “Targeted ultrasonic contrast agents for molecular imaging therapy: a brief review” Medicamundi, vol. 47, no.1, pp. 66-73, April 2003) and are known as targeted microbubbles. In these cases, safe detection of single microbubbles or small microbubble populations is highly important.
The method of detecting the presence and position of single contrast agent microbubbles or small microbubble populations is even more important when said method is provided in combination with said microbubbles having a structure capable of selectively bonding to certain predetermined types of tissues, and being further able to carry medicaments therein. In this case, microbubbles reach the predetermined tissues and bond to them and, once they are detected, they may be caused to break by using ultrasonic transmit pulses having such a strength as to generate high acoustic pressures. The destruction of microbubbles allows local drug delivery to the predetermined tissues.
Therefore, the method of detecting single microbubbles or small microbubble populations is provided, according to this invention, in combination with contrast agents whose microbubbles contain bioconjugate ligands capable of bonding to predetermined tissue types.
Also, the method of this invention is provided in combination with microbubbles containing bioconjugate ligands with drugs therein.
Therefore, the invention relates to a method for local drug administration to predetermined tissues, which provides microbubbles having a structure with bioconjugate ligands capable of selectively bonding to predetermined tissue types, and which microbubbles carry predetermnined doses of a drug therein whereas detection of microbubbles in predetermined tissues is effected by using the method of detection of single microbubbles or small microbubble populations according to this invention and according to any one of the variants as described above whereas, once the presence and/or position of single microbubbles or a small microbubble population are detected, one or more transmit pulses are transmitted, which have such a strength as to generate a sufficient acoustic pressure to destroy (rupture) said microbubbles.
Further improvements of the inventive method will form the subject of the dependent claims.